Rosmarinus officinalis L. is a species that is widely known for its culinary and medicinal uses. The purpose of the present study consisted of the evaluation of the antiproliferative and antimicrobial effects of R. officinalis-loaded liposomes (L-R). Characterization of the liposomes was performed by establishing specific parameters. The load of the obtained liposomes was analyzed using an LC-MS method, and antiproliferative assays evaluated the cell viability on a liver adenocarcinoma cell line and on a human hepatic stellate cell line. Antimicrobial assays were performed by agar–well diffusion and by broth microdilution assays. The obtained liposomes showed high encapsulation efficiency, suitable particle size, and good stability. High amounts of caffeic (81.07 ± 0.76), chlorogenic (14.10 ± 0.12), carnosic (20.03 ± 0.16), rosmarinic (39.81 ± 0.35), and ellagic (880.02 ± 0.14) acids were found in their composition, together with other polyphenols. Viability and apoptosis assays showed an intense effect on the cancerous cell line and a totally different pattern on the normal cells, indicating a selective toxicity towards the cancerous ones and an anti-proliferative mechanism. Antimicrobial potential was noticed against all tested bacteria, with a better efficacy towards Gram-positive species. These results further confirm the biological activities of R. officinalis leaf extract, and proposes and characterizes novel delivery systems for their encapsulation, enhancing the biological activities of polyphenols, and overcoming their limitations. Rosmarinus officinalis L. (rosemary) is a species belonging to the Lamiaceae family, which is known for its medicinal and culinary uses. It is widespread in the Mediterranean region, where it was first identified in the temperate climate [R. officinalis belong to the class of phenolic diterpenes (carnosic acid and carnosol) and to the class of phenolic acids (rosmarinic acid) [ climate ,2. It is climate ,3,4,5,6. climate . The veg climate ,8,9, whi climate ,4,10,11. climate . The bio climate ,8,9,13.  climate . The spe climate , which d climate ,16 or th climate ,18,19. T climate , and esp climate . The pheic acid) . They aric acid) ,23.The phenolic compounds’ role in different biological activities are limited due to low oral bioavailability in relationship with the chemical structure, molecular size, degree of polymerization, and water solubility, and these limitations may be exceeded by their incorporation into different drug-delivery systems, of which liposomes seem to be the most adequate .R. officinalis and are used to achieve more efficacy in overcoming the limitations of its phytoformulations (related to bioavailability and solubility) and to enhance its therapeutical activities [R. officinalis extract [R. officinalis extract in liposomal carriers, it was proven that these formulations may improve the limitation of using polyphenols. Existing studies cite better penetration enhancement for topical delivery [Nano-phytomedicines represent a category of drug formulations that result from a combination of nanotechnology and herbal medicine . They artivities ,30. Lipotivities ,29,30. A extract  or to en extract . Throughdelivery , a more delivery , an incrdelivery , compareR. officinalis leaf extract and testing its antimicrobial and antiproliferative potential. The novelty of the present study consists of the fact that, to the best of our knowledge, it is the only study that proposes and characterizes special delivery systems such as liposomes for the encapsulation of R. officinalis leaf extract, with the final purpose of enhancing the biological activities of its polyphenols, such as the antimicrobial and cytotoxic ones. The developed nanoformulations may represent innovative and specific vectors for the amelioration of the limitations of polyphenols, preserving or enhancing the biological properties of R. officinalis leaf extracts.With all of this taken into consideration, the aim of the present study consisted of the investigation of the encapsulation into liposomes of an The results obtained for the characterization of empty liposomes (L-E), those loaded with doxorubicin (L-DOX), and the R. officinalis leaf extract (L-R)  highlighLC-MS analysis of L-R showed significant amounts of polyphenols . The com™), derived from the ascitic fluid of a patient with adenocarcinoma of the liver, and LX-2, a human hepatic stellate cell line. The MTT assay was performed to evaluate cytotoxicity. Results were correlated with the ones obtained in the apoptosis assays. The viability and apoptosis assays showed different behavior of normal and tumoral cells exposed to the loaded liposomes. The performed analysis indicated relevant in vitro cytotoxic activity for all tested samples as well as differences related to doses and cell lines. The obtained results are presented in The in vitro antiproliferative potential of the L-R and L-DOX was assessed on two selected human cell lines, SK-Hep-1 (HTB-52The results pointed out relevant in vitro cytotoxic activity expressed by L-R against the SK-Hep-1 (HTB-52™), with the most intense antiproliferative effect observed in the case of the highest tested concentration—C5 (282.5 µM GAE/mL). The concentration of L-R that required the SK-Hep 1 cell viability to be reduced by 50% (IC50) was 181.29 ± 6.14 GAE/mL.™) and LX- 2 cells were exposed to different concentrations of L-R and L-DOX for 24 h and apoptosis was measured by the Annexin V-FITC and Ethidium homodimer III assay. The results are shown in The SK-Hep-1 (HTB-52Apoptosis assay showed that the treatment with L-R and L-DOX induced apoptosis and necrosis at varying rates in SK-Hep 1 and LX-2 cells compared to the control. The pattern of cell inhibition was different in the two cell lines and depended on the concentration of tested substance. The addition of L-R (C1-C5) to the SK-Hep 1 cell culture induced cellular death effect following 24 h compared to untreated cells. The percentages of the viable cells induced by L-R C3, C4, and C5  were similar to those determined by the highest tested concentration of doxorubicin, the positive control. The cell death mechanism was predominantly apoptosis, but in C4 it was also necrosis, with the highest percentage of necrotic cells (17.8%). After treatment with C4, the apoptotic population was 3.8%, followed by a significant increase in apoptosis rate, correlated with the concentration of L-R. Compared with other concentrations, the cells treated with C5 presented the highest rates of apoptosis, at 20.9% and 29.2%, respectively, and cell necrosis was 9.45%.R. officinalis may be a fairly viable alternative.The pattern of cellular inhibition obtained in the case of L-DOX was different compared to L-R. The percentage of cell viability was slightly decreased, but was still maintained at over 80% in C1–C3, with a higher decrease in C4 (74.2%) and especially in C5, where the percentage of cell viability was 46.3% and the apoptotic cells population was 47.7%. All data are correlated with the results obtained in the MTT test, in which the same pattern of cell viability decrease was noticed. The L-R cytotoxicity was noticed only towards the tumoral cell line, whereas on the normal cell line  the percentages of viable cells was within the range. Although the same liposomes did not exhibit a similar effect in the LX-2 cell line, L-R-induced apoptosis rates were significantly lower on the LX-2 cells, demonstrating selective toxicity of L-R. Regarding the effect of L-DOX in LX-2 cells, the average rates of apoptosis recorded was 26.42 ± 2.73%. To enhance apoptotic level, co-delivery of Doxorubicin and Results of the in vitro antimicrobial activity evaluation are presented in Bacillus cereus > MSSA > MRSA > Enterococcus faecalis) compared to the Gram-negative ones . Compared to the positive control gentamicin, these values were found to be significantly (p < 0.05) higher in case of Bacillus cereus, MSSA, MRSA, and Enterococcus faecalis. Against Enterococcus faecalis and the selected Gram-negative bacteria, L-R presented inhibitory activity similar to that of gentamicin (p > 0.05).The tested product displayed in vitro antimicrobial activity  against Similarly, the minimum inhibitory and bactericidal concentrations established using the broth microdilution method indicated better antimicrobial activity against the Gram-positive strains . ConsideR. officinalis is one of the most widely known medicinal species due to its use in the treatment of numerous pathologies. The vegetal medicinal product obtained from this species is provided by the leaves, which contain large amounts of essential oils and polyphenols. The present study aimed to bring novelty insight concerning this species by proposing novel formulations as special delivery systems. These formulations were represented by liposomes, encapsulating the leaf extract of the species, with the final purpose of overcoming the limitations of polyphenol administration.One of the most important advantages of liposomes is that they can incorporate both lipophilic and hydrophilic compounds, making them suitable candidates for the encapsulation and delivery of plant extracts . Yet, acPhytochemical analysis of liposomes showed significant amounts of phenolic acids, with high contents of ellagic, rosmarinic, caffeic, and chlorogenic acids. Carnosic acid and carnosol were the majority diterpenic phenols, and flavonoids were represented by apigenin, chrysin, rutoside, hyperoside, and luteolin-7-O-glucoside. The obtained results provide further arguments that confirm the presence of these compounds in the composition of the species, especially rosmarinic, caffeic, and chlorogenic acids, which were previously identified ,12,23,39™), a cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver) and on a human hepatic stellate cell line (LX-2), as there are no previous studies concerning liposomes loaded with rosemary-leaf extract on the two cell lines selected for the study. The choice of the two cell lines is directly related to previous reports on hepatoprotection performed by our team. This represents one of the most important aspects of novelty of the present study, together with the obtention of liposomes as special delivery systems for polyphenols. Moreover, the present study represents the first to test the antiproliferative potential of these delivery systems. The cytotoxicity and apoptosis analysis showed a different comportment of normal and tumor cells treated with L-R and L-DOX. The treatment of SK-Hep-1 cells to L-R induced a significant decrease in cell viability, and this decrease was influenced by the concentration of loaded samples (up to 169.6 µM GAE/mL). The reduction of cell viability was dose dependent; the lowest viability was identified in cells treated with the highest concentration of natural extract (C5), where the mean viability was 46.68 ± 1.32%. Similar behavior was also observed in cells treated with L-DOX, stating that the decrease in cell viability was not as high (mean viability—54.44 ± 1.92%). According to the apoptosis assay results, the decreased viability was associated with increased percentages of apoptotic and necrotic cells. The predominant mechanism of cell death induced by L-R extract in Sk-Hep-1 was therefore related to apoptosis. The cytotoxic potential was also assessed by calculating the required concentration that inhibited 50% of the Sk-Hep-1 cell line (IC50 in µM/mL). The concentration of L-R required to reduce Sk-Hep-1 cell viability by 50% was 181.29 ± 6.14 μM/mL at 24 h. For L-DOX IC50 it was 10.59 ± 0,40 µM/mL. The mechanism of protection still needs further investigation, but offers a good background for novel insights.Regarding the antiproliferative assays, to the best of our knowledge, the results obtained in the present study for the antiproliferative assays are the first from a liver adenocarcinoma cell line  to inhibit bacterial strains [Bacillus cereus, and Enterococcus faecalis. A relevant finding of this study is the inhibitory and bactericidal activity displayed against the MRSA reference strain. MRSA is recognized worldwide as a well-known prototype of multidrug resistance, and alternatives to classical antimicrobial agents are needed [Several studies documented the ability of distinct  strains ,41,42. E strains ,20 and v strains ,38. The e needed . L-R deme needed . FurtherEgg phospholipids with 80% phosphatidylcholine (Lipoid E80) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) were purchased from Lipoid GmbH , cholesterol (CHO) from sheep wool, and doxorubicin hydrochloride (DOX) and Folin-Ciocalteu reagent from Merck KgaA . Minimum Essential Medium Eagle (MEM), Dulbecco’s modified Eagle’s medium , Antibiotic-Antimycotic 100×, and 1% L-glutamine were purchased from Merck KgaA , and 10% fetal bovine serum from EuroClone . 3--2,5-diphenyl tetrazolium bromide was purchased from Merck KgaA , and dimethyl sulfoxide solution (DMSO) from Fluka . Bacterial reference strains were obtained from Oxoid Ltd. , whereas the culture mediums, Mueller Hinton Broth and Mueller Hinton agar, were purchased from Merck . All the other solvents and reagents used for analysis were of analytic-grade purity and were purchased from Merck KgaA . Reference compounds used in the LS-MS method were also of analytic-grade purity and were purchased from Phytolab, .Rosmarinus officinalis obtained from the ecological culture of PlantExtrakt . Vegetal material was identified by Lecturer Irina Ielciu, PhD, and voucher specimens were deposited at the herbarium of the Pharmaceutical Botany Department of the Faculty of Pharmacy Cluj-Napoca (Vouchers no. RO122.1-3). Air-dried leaves were ground using a Grindomix GM 200 knife mill  and subsequently macerated for 10 days with 70% v/v ethanol in water and 2–3 shakes/day and filtered at the end of the maceration period [The vegetal material consisted of the leaves of n period . For then period . The humn period .R. officinalis (L-R)-leaf extract were prepared using a modified method of the reverse-phase evaporation technique reported by Machado et al. [R. officinalis extract  and a specific amount of double-distilled water  were added over the ethanolic solution. The final mixture was stirred for 5 min at 400 rpm, after which the ethanol was evaporated under pressure at 45 °C using the rotavapor. The liposomal dispersion was downsized using a water-bath sonicator for 15 min, and the purification step was performed via centrifugation at 5000 rpm for 15 min. The supernatant was collected and used in the subsequent experiments. The same technique was applied to prepare the empty liposomes (L-E) without adding the plant extract. Equal volumes of ethanol and double-distilled water were used to prepare the L-E.The liposomes loaded with o et al. . FirstlyLiposomes loaded with DOX (L-DOX) were prepared using the active-loading method with ammonium sulfate ,48. To tv/v/v), and the solution was treated with Folin–Ciocalteu reagent diluted in water (1:10 v/v) and an aqueous solution of Na₂CO₃ 7.5%, according to the previously mentioned protocol. The absorbance of the supernatant was measured at 740 nm using a UV-VIS spectrophotometer .The quantification of the polyphenolic content of L-R was performed using the colorimetric reaction with Folin–Ciocalteu reagent as reported by Postescu et al. . In thisv/v), the measurements were performed. The equipment used for DOX quantification was an Agilent 1100 Series HPLC system  and a Zorbax C18 column (3.5 μm) . The quantification method involved a gradient elution with acetonitrile and formic acid 0.1%. The DOX retention time was 0.95 min.DOX encapsulated in liposomes was quantified using an HPLC-validated method with UV detection . After dThe encapsulation efficiency (EE%) of the liposomes was calculated using the following formula:v/v), and each sample was analyzed in triplicate.Particle size and polydispersity index (PdI) were determined by the dynamic light-scattering method, and zeta potential by laser Doppler electrophoresis using a Zetasizer NanoZS analyzer . Prior to these measurements, the liposomes were diluted in double-distilled water 1:100  could be identified. Identified and quantified compounds can be found in The LC/MS analysis was carried out on a Shimadzu Nexera I LC/MS-8045  UHPLC system equipped with a quaternary pump, an autosampler, an ESI probe, and a Quadrupole rod mass spectrometer. Separation was obtained using a Luna C18 reversed-phase column  purchased from Phenomenex . The column temperature was set to 40 °C during the whole duration of the analysis. The mobile phase  consisteDetection was carried out on a quadrupole rod mass spectrometer, with electrospray ionization (ESI), in both negative and positive multiple-reaction-monitoring (MRM) ion modes. 2 = 0.9964–0.9999) for the quantification of the compounds and references. The present method was validated by assessing linearity, precision, and accuracy according to International Conference on Harmonization (ICH) guidelines. LOD and LOQ were calculated after injecting a series of different concentrations for each analyzed sample. Extracts were evaluated for precision under optimized conditions. The method’s accuracy was determined in duplicate with a recovery experiment. All samples were injected in triplicate. Results obtained during validation can be found in The injection volume was 1 μL for each reference and was maintained at each concentration. Identification was performed by comparison of the retention times, MS spectra, and the transitions between the separated compounds and references. Identification and quantification were performed based on the main transition from the MS spectra of each compound. Calibration curves were determined (R™) cell line derived from the ascitic fluid of a patient with adenocarcinoma of the liver [™) cells were maintained in Minimum Essential Medium Eagle (MEM) supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic 100× and 1% L-glutamine in a 5% CO2 incubator  at 37 °C in a humidified atmosphere, according to standard conditions. The LX-2 cell line was maintained in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum and 1% Antibiotic-Antimycotic 100× and 1% L-glutamine [The cytotoxicity assay of L-R and L-DOX was performed using the SK-Hep-1 , of doxorubicin (positive control)  and of the loaded liposomes were added. The concentrations were determined according to the amount of total polyphenols  in the L-R and L-DOX, respectively. Other controls were represented by untreated cells (negative control) and cells treated with L-E . Following 24 h incubation at 37 °C in a humidified atmosphere with 5% CO2, the medium was removed and 100 µL of 1 mg/mL MTT solution was added to determine the viability of the cells. After 3 h of incubation at 37 °C in dark, the MTT solution was removed from each well and 150 µL of dimethyl sulfoxide solution (DMSO) was added. Spectrophotometric readings at 450 nm were performed with a BioTek Synergy 2 microplate reader . The cell viability percentages (%) were determined based on the absorbance ratio between cell cultures treated with L-R and L-DOX and the negative controls (untreated cells) multiplied by 100. The concentrations required to inhibit 50% of cell proliferation  for L-R and L-DOX were calculated from the dose–response curve obtained using non-linear regression. Each experiment was performed in triplicate [The cytotoxicity of L-R and L-DOX was assessed using the -2,5-diphenyl tetrazolium bromide (MTT) assay according to a previously published protocol. Briefly, for each of the selected cell lines, 1 × 10iplicate ,53,54.Apoptosis was assessed with the Apoptosis & Necrosis Quantification Kit . Cells were stained with Annexin V-FITC and Ethidium Homodimer III (EthD-III) according to the kit’s instructions, and the fluorescent intensity was read with a BD FACS Canto II flow cytometer  with a two-laser configuration: 20 mW argon solid state at 488 nm, and 17 mW neon-helium (NeHe) at 633 nm. The instrument was set to acquire information from the corresponding photodetector for Annexin V (FL1-A) and EthD-III (FL3-A) on a logarithmic scale. An unstained control sample was analyzed for FSC-A (forward scatter) and SSC-A (side scatter) signals in order to identify the cell population of interest and remove debris. The fluid pressure was set to a minimum (low) so the acquisition speed was appropriate. Subsequently, the stained samples were read. Fluorescence detection was achieved with the 488 nm laser and 525/50 filter for Annexin V and the 695/40 filter for EthD-III. A total of 10,000 events (cells) was run for each tube. Analysis was performed using the BD FACSDiva 6.1.2 software. Fluorescent intensity was shown in dot plots, divided into 4 quadrants each. Cells that did not bind any of the fluorochromes appeared in quadrant 3 (Q3—viable cells). Cells that were only stained with Annexin V-FITC appeared in quadrant 1 (Q1—apoptotic cells), whereas cells that were stained with both Annexin and EthD-III appeared in quadrant 2 (Q2—late apoptotic cells). Cells in quadrant 4 (Q4) were only stained with EthD-III and were necrotic cells. The percentages of apoptotic cells were calculated from Q2 and Q3. A total of 10,000 cells was analyzed and included in each dot plot.To investigate the in vitro antimicrobial activity of the L-R, several methods were selected. The initial screening was based on the agar well-diffusion assay, a modified EUCAST   disk-difStaphylococcus aureus ATCC 25923 , Staphylococcus aureus ATCC 700699 , Bacillus cereus ATCC 14579, Enterococcus faecalis ATCC 29219, Escherichia coli ATCC 25922, Salmonella enterica serovar Typhimurium ATCC 14028, and Salmonella enterica serovar Enteritidis ATCC 13076. For each organism, an inoculum was made by suspending pure culture in Mueller Hinton (MH) broth for 24 h to obtain 10−6 colony-forming units (CFU)/mL according to the McFarland scale. The MH agar plate surface was “flood-inoculated” with the bacterial inoculum and prepared for extract evaluation; 6 m-diameter wells (three for each sample) were aseptically made in the MH agar to contain 60 μL of the tested product and 70% ethanol in water v/v (as the negative control). Gentamicin was also included as standard antibiotic. The growth-inhibition-zone diameters in millimeters were measured after 24 h of incubation at 37 °C. Minimum inhibitory (MIC) and bactericidal (MBC) concentrations were determined by a broth-microdilution method. Two-fold serial dilutions were made in 100 µL broth; 5.0 µL of a 24 h 10−6 CFU/mL bacterial inoculum were added to each well and incubated for 24 h at 37 °C. MIC values were read as the lowest concentrations able to inhibit the visible growth of bacteria (no turbidity in the well) compared to the negative control (broth). From each well, 10.0 µL were cultured on MH agar plates for 24 h at 37 °C. MBC values were read as the lowest concentrations associated with no visible bacterial growth on the agar plates. All these tests were performed in triplicate. Based on the MBC/MIC ratio, the MIC index was also calculated for each sample to evaluate whether the extract exhibited bactericidal (MBC/MIC ≤ 4) or bacteriostatic (MBC/MIC > 4) effect against the tested bacterial strains [Seven reference strains were included:  strains .p-value lower than 0.05 was considered statistically significant [All statistical analyses were performed using ANOVA GraphPad Prism software version 6.0 . The obtained results are expressed as the mean ± standard deviation (SD). One-way analysis of variance (ANOVA) was used, followed by Tukey’s post hoc test, to determine statistical significance. A nificant .R. officinalis is a widely known species with numerous therapeutic uses that are related to its polyphenols and essential oils. The present study provides novel and original insight into the species by proposing special delivery systems, liposomes encapsulating the leaf extract, for the evaluation of its antimicrobial and antiproliferative effects due to polyphenols. Not only were these biological activities confirmed, but novel insights on them and on their mechanism of action were also revealed. The results obtained therefore represent an important basis for further studies of innovative delivery systems loaded with different products of rosemary, which could be used for the treatment of numerous diseases as different pharmaceutical formulations. Moreover, the present study raises important arguments to offer a solid scientific background to sustain their use in the treatment of hepatic-related pathologies or antimicrobial ones. In this way, important perspectives for future studies on the species and its formulations are open in order to further elucidate mechanisms of action or to confirm the biological activity of the species.


Specify the risk rate of incorrect patient classification based on the night-to-day ratio specification from singular 24-h ABPM in comparison to the results of 7-day ABPM monitoring  1197 24 h cycles were enrolled in 171 subjects in the study and divided into 4 groups: group 1 , group 2 , group 3 (40 patients with ischemic coronary artery disease without exercise), and group 4 (51 patients with ischemic coronary artery disease following cardiovascular rehabilitation). The subject of the evaluation was the percentage rate of incorrect subject classification  based on the mean blood pressure values for 7 days and from seven independent 24-hour cycles . p < 0.594; 56% vs. 54%) or physical activity  of the monitored individuals.  In the case of the individuals included in the monitored groups, the mean night-to-day ratio-based  classification accordance ranged between 59% and 62%. Only in singular cases did the accordance reach 0% or 100%. The accordance size was not dependent on the health or cardiovascular disease . Although blood pressure measurement in outpatient care is standardised and governed by accurately defined principles, there is a statistically significant probability of erroneous hypertension diagnosis , 2. OtheMost authors base the previous classification according to the night-to-day ratio solely on 24-hour blood pressure monitoring. Night-to-day ratio may be specified by evaluating the mean blood pressure values from the seven monitored days as well as by evaluating the individual 24-hour cycles . The purIn total, 1197 24-h cycles were enrolled in 171 subjects in the study and divided into 4 groups. The first group included 40 subjects (20 men and 20 women)  without chronic medication. The second group of healthy exercise-training individuals included 20 men and 20 women (mean age 29 ± 7.8 yrs and mean BMI 22.3 ± 5.18) without chronic medication. The third group included 40 patients with chronic ischemic coronary artery disease . The fourth group included 51 patients with chronic ischemic coronary artery disease  following cardiovascular rehabilitation. The first part of the study involved evaluating the 40 healthy individuals of the first group without chronic medication and 40 patients (group 3) with chronic ischemic coronary artery disease without exercise. The second part of the study focused on assessing night-to-day ratio changes in the healthy individuals following controlled combined aerobic-resistance exercise (group 2) and in the patients with chronic ischemic coronary artery disease (group 4) following cardiovascular rehabilitation. All patients (third and fourth groups) were treated with beta-blockers, ACE inhibitors, and statins. They were symptomatically stable and their medication was not changed during rehabilitation or during ambulatory monitoring.The ambulatory monitoring (7-day ABPM) was performed with ambulatory blood pressure monitoring devices TM–2430  based on both the cuff-oscillometric and Korotkoff sound method. The device automatically recorded all the regular measurements for the period of continuous seven days, between 6:00 a.m. and 10:00 p.m. every 30 minutes and between 10:00 p.m. and 6:00 a.m. every 60 minutes. Such short intervals are necessary to obtain a sufficient number of representative measurements. The monitored individuals recorded their daily activities, their sleep times, their subjective problems, and their pharmaceutical intakes throughout the monitoring period. To make the blood pressure values objectively comparable for all tested individuals, the time interval in which the blood pressure values corresponded to the sleep/awake times needed to be defined. For that purpose, the mean nocturnal blood pressure value for individual days was calculated for the time interval between 1:00 and 6:00 a.m., and the daily blood pressure means for the time interval between 10:00 a.m. and 10:00 p.m. Blood pressure values for the periods between 7:00 and 9:00 a.m. and 10:00 and 12:00 p.m. were excluded from night-to-day ratio calculation for the purpose of data standardisation. This way, the time intervals for blood pressure recording corresponding to the night sleep and day awakening periods were unified for all individuals enrolled.max). The minimum time span between the workouts was 24 hours. The workout consisted of a 10-minute warm-up, 25 minutes of aerobic exercise (spinning bicycle or bicycle ergometer), 15 minutes of resistance exercise on gym machines  with 40–50% 1-RM (one repetition maximum) intensity (3 series of 10 exercises), and a 10-minute cool-down (stretching of the main muscle groups). The cardiovascular exercise (group 4) took the form of sixty-minute workouts scheduled two to three times weekly. The nature of the workouts was similar, combined aerobic-resistance exercise. The workout intensity was set at the level of the first ventilation threshold (VAT-1), 666 corresponding to the following workout parameters: load (W) and heart rate (HR) [Combined aerobic-resistance exercise for 40 healthy individuals (group 2) was scheduled twice weekly in the form of 60-minute workouts with a load intensity at the level of submaximum heart frequency (75–85% SFate (HR) . The resday − SBPnight)/SBPday [t-test assuming unequal variances was used. P value = 0.05 was considered as statistically significant. Differences between the percentage of accordance of the overall mean for 7 days and the percentage of accordance of the mode were tested by paired t-test.The arithmetic mean was calculated for every hour from all measured systolic blood pressure (SBP) and diastolic blood pressure (DBP) values obtained by the 7-day monitoring, plus weighed means from the individual 24-hour cycles and the whole 7-day interval. The night-to-day ratio was calculated using the following formula: 100 × (SBP)/SBPday . Each stp < 0.0017, 62% vs. 55%).p < 0.062).p < 0.007, 59% vs. 53%).p < 0.004).1p < 0.833; 55% vs. 54%). The difference in the accordance size between young healthy individuals . The difference in the accordance size between patients without exercise .The difference in the accordance size between the exercising subjects  Tables  and 4 anexercise  and patiexercise  was not p < 0.818; 55% vs. 56%) in the night-to-day ratio-based categorisation accordance rate as a consequence of the performed physical activity.In addition, in the case of the comparison between the group of healthy individuals without physical activity  and the There are studies pointing to ethnic differences in blood pressure response to physical activity –19. LikeFurthermore, the next part of the study evaluating healthy subjects with controlled physical activity  found thInsufficient blood pressure decrease during night sleep (nondippers) is certainly a significant predictive factor for mortality, but other coincident risk factors  must also be taken into account , 29, 30.The same applies to risk groups, i.e., seniors, diabetic patients, obese individuals, or pregnant women , 33. The1Night-to-day ratio specification for each day from 7-day ABPM also represents a doubtless advantage in that it allows for the recording of individual variability and frequency of occurrence of high-risk days. Discussions with the examined individuals can then find which particular activity of the subject caused a blood pressure response of the nondipper (ND), extreme dipper (ED), or reverse dipper (R) type.Although the relationships between circadian blood pressure variability and physical activity are not yet quite clear, most studies dealing with this issue point to the positive effects of physical activity on the cardiovascular system and blood pressure values , 38.According to the results of our study combined training does not change the variability of blood pressure .Due to lifestyle in the so-called “civilized” world, the percentage of the urban population with blood pressure higher than 140/90 mmHg or who uses antihypertensive drugs increases with age. The high prevalence of cardiovascular risk factors in populations is significantly associated with life conditions. Abnormal dipping patterns is important prognostic markers for future cardiovascular morbidity in resistant hypertension patients. Extreme dipping is a protective factor in younger individuals but a hazardous factor in the elderly and in individuals with previous cardiovascular diseases . High caIn contrast, the findings in the isolated populations  showed that neither blood pressure elevation with age is inevitable, nor is the high prevalence of hypertension .On the basis of the obtained results, the following procedure can be recommended for clinical practice. If the standard 24-hour monitoring is used, then obtain at least 3 mutually independent 24-hour cycle data for every examined individual. If at least two of them result in the same night-to-day ratio and the third night-to-day ratio is only slightly different, the thus obtained index can be considered trustworthy. Otherwise, the correct night-to-day ratio can only be obtained by 7-day ABPM and, in addition, the resulting night-to-day ratio calculated from the mean values obtained in the course of the 7 days must be compared to the result modes obtained for the individual monitoring days. Our own clinical experience teaches us that there are subjects whose accurate night-to-day ratio-based classification is difficult due to the high-value variability between days. Therefore, the conclusion can be drawn that the mean value of the night-to-day ratio found by ambulatory BP monitoring across 7 days is a much more accurate method of valid value specification when compared to 24-h ABPM. In the case of the individuals included in the monitored groups, the mean night-to-day ratio-based  classification accordance ranged between 59% and 62%. Only in singular cases did the mean accordance reach 100%. The accordance size was not dependent on the gender, age, health, or cardiovascular disease, or physical activity of the monitored individuals.


Natural products are currently attracting more focus due to structural diversity and safety in contrast to synthetic drugs. In the present work, computational studies were conducted on natural product-like caffeine derivatives to search for novel potent candidates acting as dual MAO-B inhibitors/AA2AR antagonists for Parkinson’s disease. Our findings revealed two natural products among the top hits: CNP0202316 and CNP0365210 fulfill the requirements of drugs acting on the brain. The selected lead compounds were further studied using molecular dynamics simulation to assess their stability with MAO-B. Current findings might shift the interest towards natural-based compounds and could be exploited to further optimize caffeine derivatives into a successful dual-target-directed drug for managing and halting the neuronal damage in Parkinson’s disease patients.Parkinson’s disease is considered the second most frequent neurodegenerative disease. It is described by the loss of dopaminergic neurons in the mid-brain. For many decades, L-DOPA has been considered as the gold standard for treating Parkinson’s disease motor symptoms, however, due to the decrease of efficacy, in the long run, there is an urgent need for novel antiparkinsonian drugs. Caffeine derivatives have been reported several times for their neuroprotective properties and dual blockade of monoamine oxidase (MAO) and adenosine A Neurodeagonists .Monoamine Oxidase (MAO) (EC 1.4.3.4) belongs to a family of flavin adenine dinucleotide (FAD)-dependant enzymes that are expressed in the outer mitochondrial membrane of neuronal cells. The MAO enzymes are responsible for the oxidative deamination of monoamine neurotransmitters such as dopamine, adrenaline, and noradrenaline in the central nervous system (CNS) , 7. The MAO-A preferentially metabolizes serotonin while MAO-B preferentially deaminates 2-phenylethylamine and benzylamine. Dopamine, norepinephrine, and epinephrine are substrates of both isoforms in most animal tissues .2O2) inducing oxidative damage and apoptotic signaling events [During aging, the expression of MAO-B increases in the brain and is connected with an enhanced dopamine metabolism which results in an increased reactive oxygen species (ROS) production such as hydrogen peroxide  which encourages further research on new substitutions to the caffeine core [Previously approved MAO-B inhibitors are selegiline and rasagiline which irreversibly inhibit MAO-B with an ICectively . The latf 450 nM . Istrade nM [2AR . Howeverine core . The che3 volume while the crystal structure of MAO-B contains a bipartite cavity structure with an entrance cavity of ∼290 Å3 and a substrate cavity of ∼400 Å3 [The crystal structure of MAO-A (PDB ID: 2Z5Y) has a monopartite substrate cavity of ∼550 Å ∼400 Å3 . ILE-199 ∼400 Å3 , 19. TheThe structural study revealed that MAO-B (PDB ID: 2V5Z) is formed by two monomers consisting of a globular domain anchored to the membrane through a C-terminal helix . MAO-B a2AR antagonists. Pretorius et al. synthesized a series of C-8 substituted caffeinyl analogues and it was found that the compound bearing a 4-phenylbutadien moiety is the most potent candidate for MAO-B and AA2AR [2AR antagonism, whereas replacements occurring at C1 and C3 are optimal for AA2AR but not detrimental for MAO-B [There is a great deal of literature supporting the use and efficacy of natural products (NP) in PD such as flavonoids, xanthones, phenolic derivatives, alkaloids, and caffeine , 22. Thend AA2AR . On the or MAO-B . Althougor MAO-B .NPs and NP-based compounds are an ideal choice for scientists and researchers due to the broad-spectrum activity of NPs with their minimal or no toxic effect on human health . The lit2AR, a validated target for PD [2AR. ADMET properties were evaluated using in silico methods. Finally, molecular dynamics simulations were performed to study the interactions and the stability between the selected compounds and MAO-B over the simulation time.In the present study, a substructure search was conducted on natural products databases to retrieve caffeine-containing natural products since it is known for its neuroprotective properties and its potency to act as an antagonist of AAt for PD . Structu22.1https://coconut.naturalproducts.net/); the largest open-source natural products (NP) database to date containing more than 400 000 unique NP from over 50 sources [To retrieve all the available natural compounds based on the caffeine scaffold, we used the COCONUT database  in complex with caffeine were retrieved from the RCSB Protein Data Bank (https://www.rcsb.org/) [2AR in x, y, and z directions, respectively. Lastly, the generated coordinates for the grid box were saved in a text file.Crystal structure of MAO-B  in complex with safinamide and crystal structure of AAsb.org/) . Residuesb.org/) . Water msb.org/) . Finallysb.org/) . The gri2.3The selected caffeine derivatives were split into multiple files, with each file containing a single ligand. The 3D conformations were generated for all the compounds, geometrical optimization was performed using Merck molecular force field (MMFF94) implemented in the Open Babel chemical toolbox . The min2.4Structure-based virtual screening was performed using a Perl script for the automated execution of AutoDock Vina 1.1.2 . The pro2.5The ligands were ranked by their binding affinities. The compounds displaying a binding score of −8.0 kcal/mol or less were subject to further analysis. The conformations of the selected compounds were visualized using UCSF Chimera visualization software and superposed to the ligand of reference . Discove2.6http://www.swissadme.ch/) [https://tox-new.charite.de/protox_II/) [The profiling of compound pharmacokinetics is very essential in drug discovery. As of today, many online tools are available to predict the ADMET profiles of drugs based on their chemical properties . In silidme.ch/) . Lipinskdme.ch/) . Other pdme.ch/) . Hepatottox_II/) .2.7AMBER16 software  was empl2.8Gbinding) of the investigated inhibitors in complex with MAO-B [Gbinding) binding energy was calculated by the standard formula:The molecular mechanics-generalized Born surface area (MM-GBSA) approach was applied to estimate the binding free energies  [Molecular docking protocol was first validated by cross-docking the co-crystallized ligands using the same parameters that were applied for the studied compounds against different crystallographic structures (PDB ID: 2V61 for MAO-B and PDB ID: 5IU4 for AA2AR) , 58. Com50) for MAO-B and dissociation constants (Ki) for AA2AR respectively [2AR, which confirms the reliability of the molecular docking approach to study the natural product-like caffeine derivatives with MAO-B. The correlation charts, correlation coefficients, and slopes are shown in Additionally, molecular docking accuracy was further validated using two datasets of 10 caffeine derivatives with reported half-maximal inhibitory concentrations . The highest-ranking molecules displaying a docking score of −8.0 kcal/mol or less were further analyzed based on their interactions with the MAO-B active site cavity. Molecular docking results and protein-ligand interactions are shown in Table 1.3.3In silico pharmacokinetics, toxicity, and drug-likeness prediction results are shown in 3.4According to the molecular docking and ADMET-based screening as summarized in Safinamide was found to interact through hydrogen bonding with GLN-206 which is known to be a hydrogen bond acceptor for the majority of MAO-B inhibitors and is by the literature . StructuIt has been shown in previous studies that hydrophobic interactions through the phenyl ring are vital for establishing MAO-B binding and are more favorable than all other interactions such as hydrogen or halogen bonds . However2AR to assess their binding affinities with the AA2AR active site and compare their binding conformations to istradefylline. Molecular docking results and protein-ligand interactions are shown in Alternatively, the selected compounds were analyzed through molecular docking studies with AA2AR antagonists [The binding poses of the selected compounds and istradefylline were superposed to the co-crystallized structure of caffeine . The stragonists . A large3.5Gbinding) and are illustrated in Gbinding = −37.9 kcal/mol). The comparison of safinamide with CNP0202316 and CNP0365210 unveiled competing for binding affinities proposing the in silico prospectivity of the two molecules as MAO-B inhibitors.Molecular dynamics (MD) simulations were applied to probe the stability of the selected ligand-protein complexes, structural specifics, conformational flexibilities, and realize reliable inhibitor-enzyme binding affinities , 67. TheEvdw) energy was a considerable contributor to CNP0202316, CNP0365210, and safinamide-MAO-B binding affinities with average values of −50.9, −46.8, and −47.3 kcal/mol, respectively. ΔEele was effectual with average values of −5.2, −11.1, and −25.3 kcal/mol for the CNP0202316, CNP0365210, and safinamide-MAO-B binding affinities, respectively.The calculated MM-GBSA binding energies were then decomposed into separate components to recognize the vigor in the binding of MAO-B with CNP0202316, CNP0365210, and safinamide . The vanThe binding energies of CNP0202316, CNP0365210, and safinamide in complex with MAO-B were further decomposed at the per-residue level, and the amino acid residues with free energy contribution <−0.50 kcal/mol were depicted . LEU-1713.6Molecular docking calculations, and MD simulations combined with MM-GBSA binding energy calculations, unveiled the most potent molecules as potential MAO-B inhibitors. The MD-based analysis could be required to demonstrate structural and energetic stabilities for the scrutinized inhibitors in complex with MAO-B. The structural and energetical analysis included root-mean-square deviation (RMSD), Center-of-Mass (CoM) distance, and binding energy per-frame.3.6.1The root-mean-square deviation (RMSD) values of the backbone atoms within the whole complex throughout the simulation time were estimated to monitor the structural stability of the CNP0202316, CNP0365210, and safinamide in complex with MAO-B. The RMSD of the backbone atoms as a function of time following the initial structure of the three investigated systems is displayed in 3.6.2To get a more in-depth insight into the stability of the selected compounds throughout the MD simulation time, center-of-mass (CoM) distances were evaluated . The mos3.6.3Gbinding) of −36.6, −34.5, and −37.9 kcal/mol, respectively. Based on this analysis, all investigated complexes preserved their stability over the 100 ns MD simulations.The comprehensive structural stability of CNP0202316, CNP0365210, and safinamide complexed with MAO-B was evaluated throughout a 100 ns MD simulation via inspecting the correlation between the binding energy per frame and time . Overall4Evdw) energy is a considerable contributor to the stability of MAO-B inhibitors. This finding confirms that hydrophobic interactions outweigh any other interactions in terms of MAO-B inhibition, and is also supported by the literature [2AR. The propylbenzene moiety attached to the imidazole of the caffeine in CNP0202316 at position C7 is more favorable to the hydrophobic pocket of AA2AR, whereas the oxygen atom of the xanthine core maintained a hydrogen bond with ASN-253 that is deemed crucial to the binding of most AA2AR antagonists [The results obtained from the molecular docking study with MAO-B were further analyzed through molecular dynamics simulations and binding free energy calculations. MM-GBSA binding energies further confirmed that van der Waals (Δterature . Post-MDterature , 69. Meaterature . Moreoveterature . For MAOagonists . Thus, t52AR antagonists. Structure-based virtual screening and ADMET analysis revealed two natural products that fulfill the requirements for drugs acting on the brain. The selected compounds in complex with MAO-B were subject to molecular dynamics simulations to assess their stability over the simulation time along with the inhibitor of reference, safinamide. Our findings show that the presence of the phenyl ring in the selected compounds is crucial for the ligands to fill the long-shaped cavity of the MAO-B active site and is a major contributor to various van der Waals interactions responsible for the stability and the tight-binding of these compounds to MAO-B. Similarly, the propylbenzene moiety was found to be more favorable for the hydrophobic pocket of AA2AR especially when linked at position C7 of the xanthine core which allowed the caffeine core to adopt a similar conformation to istradefylline suggesting the dual-target properties of the identified natural products. In conclusion, the structure-based virtual screening helped provide valuable insight on the studied natural product-like caffeine derivatives and our findings may attract more focus for the development of novel antiparkinsonian drugs with dual-targeting properties. However, in vitro experiments such as bioactivity assays for MAO-B and AA2AR, membrane permeability and cell viability assays remains necessary to further validate these findings.The present study aimed to find novel natural product-like caffeine derivatives as potential dual MAO-B inhibitors/AA


The alterations of glycosylation, which is a common post-translational modification of proteins, have been acknowledged as key events in breast cancer (BC) oncogenesis and progression. The aberrant expression of glycosyltransferases leads to aberrant glycosylation patterns, posing the diagnostic potential in BC outcomes. The present study aims to establish a glycosyltransferase-based signature to predict BC prognosis and response to immune checkpoint inhibitors. We firstly screened 9 glycosyltransferase genes from The Cancer Genome Atlas (TCGA) database and accordingly established a glyco-signature for predicting the prognosis in BC patients. Patients with BC were successfully divided into high-risk and low-risk groups based on the median cutoff point for risk scores in this signature. Next, the combinational analyses of univariate and multivariate Cox regression, Kaplan–Meier, and receiver operating characteristic (ROC) curves were used to prove that this glyco-signature possessed excellent predictive performance for prognosis of BC patients, as the high-risk group possessed worse outcomes, in comparison to the low-risk group. Additionally, the Gene Set Enrichment Analysis (GSEA) and immunologic infiltration analysis were adopted and indicated that there was a more immunosuppressive state in the high-risk group than that in the low-risk group. The clinical sample validation verified that glycosyltransferase genes were differentially expressed in patients in the low- and high-risk groups, while the biomarkers of antitumor M1 macrophages were increased and N-glycosyltransferase STT3A decreased in the low-risk group. The final in vitro assay showed that the silencing of STT3A suppressed the proliferation and migration of BC cells. Collectively, our well-constructed glyco-signature is able to distinguish the high- and low-risk groups and accordingly predict BC prognosis, which will synergistically promote the prognosis evaluation and provide new immunotherapeutic targets for combating BC. Breast cancer (BC) is the most prevalent malignancy and the primary cause of cancer-related deaths in women worldwide . AccordiO-glycan truncation, sialylation, fucosylation, and N-glycan branching are the most common alterations of cancer-related glycosylation, which drive several malignant behaviors of tumors, including tumor cell invasion and dissociation, angiogenesis, metastasis, immune modulation, and cell–matrix interactions (N-acetylglucosaminyl transferase (B3GNT3) participated in the PD-1/PD-L1 interaction and B3GNT3-mediated glycosylated PD-L1 suppressed T-cell activity in triple-negative BC  was correlated with dedifferentiation of hepatocellular carcinoma, intrahepatic metastasis, and poor prognosis  increased O-glycosylation of α2M to promote the migration and invasion of BC and that the high expression of GALNT6 in BC patients suggested a shorter overall survival (OS)  . It is min vitro assay proved that the selected glycosyltransferase genes were associated with the immune state and malignant behaviors of BC. Our results indicated that our model based on glycosyltransferase genes was capable of predicting the prognosis and immune state in BC patients. The detailed flowchart could be seen in Therefore, it is intriguing to explore the potential of glycosyltransferases for constructing a prognostic-predicting risk model of BC. In the present study, we firstly screened a profile of 9 differentially expressed glycosyltransferase genes depending on genomic information of 1,089 BC samples and accordingly constructed a prognostic-predicting risk signature. In accordance with the median risk score, the BC cases were successfully classified into low-risk and high-risk groups. These two groups showed distinct differences in OS, gene expression, immune infiltration, ICI response, and chemosensitivity. Meanwhile, clinical sample validation and https://portal.gdc.cancer.gov/). The detailed clinical information of included BC patients is summarized in Publicly available gene-expression data and related clinical information were obtained from The Cancer Genome Atlas (TCGA) database (The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses are online databases and were used to perform functional enrichment and pathway enrichment regarding the differently expressed glycosyltransferase genes between BC patients and normal samples with the “enrichplot” R package . The Genhttps://acgg.asia/ggdb2/). The OS of BC patients was considered and calculated for BC prognosis in the Cox regression analysis. In addition, the lasso regression was performed to further compress glycosyltransferase genes. Then, the glyco-signature for predicting the prognosis of BC patients was established using multivariate Cox regression analyses. The calculation of the risk score was based on the following formula:To ascertain prognosis-related glycosyltransferase genes, univariate Cox regression analysis of 169 glycosyltransferase genes was firstly adopted in the training set to select 13 glycosyltransferase genes related to the BC prognosis (p < 0.05). The 169 glycosyltransferase genes were extracted from GlycoGene DataBase  survival analysis using R language v4.0.2 (p < 0.05).Independent prognostic analysis and multivariate independent prognostic analysis were conducted because glycemic signatures and other clinical parameters, including age, stage, stages T, N, and M, were covariates. p-Value and hazard ratios (HRs) were displayed in the forest plots. Next, a nomogram was established depending on this glyco-signature and clinical parameters to evaluate the 1-, 3-, and 5-year OS of BC patients using the “rms” R package. Nomogram is a graphical representation of a complex mathematical formula, which could visualize the multivariate Cox regression and predict the OS of BC patients . CalibraThe mutation and copy number alteration (CNA) regarding the 9 glycosyltransferase genes were obtained through segmentation analysis and GISTIC algorithm in the cBioPortal . BesidesThe fraction scores of 22 immune cell subsets, as well as 29 pivotal pathways in the samples, were calculated using single-sample gene set enrichment analysis (ssGSEA) in the “gsva” R package. ESTIMATE algorithm was then performed to figure out the stromal score and immune score and tumor purity in the BC samples.https://discover.nci.nih.gov/cellminer/). Wilcoxon’s test was conducted to analyze the significance of the difference in IC50 between the two groups.Half of the maximum inhibitory concentration IC50 was applied to evaluate the chemoreceptive difference between the low-risk and high-risk groups. The mRNA profiles and drug sensitivity IC50 values were acquired on the CELLMINER website  was performed to obtain the total RNA of BC tissues. Then, the concentration and purity of total RNA were estimated by using a NanoDrop 2000 spectrophotometer . The RNA was reverse transcribed into complementary DNA (cDNA) using the 1st Strand cDNA Synthesis Kit . The qRT-PCR analysis in duplicate samples was carried out with SYBR Green™ Master Mix  in a QuantStudio1 PCR . Primer sequences of these 9 glycosyltransferase genes used for RT-qPCR are summarized in For immunohistochemistry (IHC), the BC tissue segments were deparaffinized, rehydrated through a graded ethanol series, and retrieved by heating slides at 100°C for 1 h in citrate buffer. The anti-hSTT3A antibodies  were applied. Next, the sections were washed in TBST solution and incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for about 1 h. The antigen–antibody complex was visualized using DAB Peroxidase Substrate Kit . The IHC images were obtained under a SOPTOP CX40 microscope (China).For immunofluorescence (IF), sections were incubated with an anti-hSTT3A antibody  at 4°C overnight. After being washed 3 times with TBST, the sections were incubated with a cocktail of secondary antibodies  for 1 h at room temperature. Nuclear 4,6-diamidino-2-phenylindole  was utilized for counterstaining the slides. Images were captured using a fluorescence microscope under the corresponding laser wavelength .3/well) with 3 wells for each group. After silencing of STT3A, the CCK-8 assay was performed by adding 10 μl of CCK-8 solution in each well, with subsequent incubation in an incubator for 2 h in a dark environment. Finally, the absorbance was analyzed at a 450-nm wavelength under a microplate reader .The proliferation capabilities of MCF-7 and MDA-MB-231 cells were estimated by using the cell counting kit-8 (CCK-8) assay . The cells were inoculated in a 96-well plate  were put in the lower cell chamber as an attractant. At 24 h post-incubation at 37°C, the methanol and 0.1% crystal violet were added to fix and stain the invaded cells in the lower chambers. Finally, the number of invaded cells was counted by ImageJ software.Transwell migration champers containing 24-well plates  were used to assess the migration ability. Firstly, a total of 5 × 10Wound healing assay was used to assess the migration of MCF-7 and MDA-MB-231 cells. After the cells confluenced to form a single cell layer, the cell monolayers were lightly scratched with the tip of a 200-μl pipette. Afterward, the cells were incubated in DMEM/F12 medium without FBS at 37°C of 24 h. The horizontal distance of migrated cells from the wound edge was calculated by ImageJ software .N-glycans in BC samples, as well as in MCF-7 and MDA-MB-231 cells. Total proteins were extracted using lysis buffer , and their concentrations were measured by bicinchoninic acid (BCA) protein assay . The equal content of extracted protein was separated by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene fluoride (PVDF) membranes . After being blocked with Carbo-Free Blocking Solution  for 30 min, the membranes were then incubated with biotinylated lectins for 30 min at room temperature, including concanavalin A (ConA), Phaseolus vulgaris Leucoagglutinin (PHA-L), and P. vulgaris erythroagglutinin (PHA-E) , which were prepared and diluted in PBS at 20 µg/ml concentration. Afterward, the PVDF membranes were incubated with HRP streptavidin  at 1:2,000 dilution for 1 h and detected by using enhanced chemiluminescence (ECL) assay kit .Lectin blot was applied to assess the expression level of The KM curve was applied to compare the OS among the two risk groups. Univariate and multivariate Cox regression analyses were applied to screen independent prognostic variables. The receiver operating characteristic (ROC) curve was employed to validate the diagnostic value of the signature. Student’s t-test was adopted to determine the relationships between the risk score and clinicopathological factors. All statistical analyses were carried out with R language R x64 4.0.5. p-Value <0.05 was regarded as statistically significant.O-glycan, N-glycan, and glycosphingolipid biosynthesis as shown in The GO and KEGG pathway analyses were utilized to reveal the possible cellular biological effects of glycosyltransferase-associated differently expressed genes (DEGs). The top 10 enriched GO terms of biological process (BP), cellular component (CC), and molecular function (MF) for the glycosyltransferase genes are described as a scatter diagram in Through Cox regression analysis, it was found that 13 differently expressed glycosyltransferase genes were associated with BC prognosis (p < 0.05) (Based on the median risk score (0.976), we divided 1,089 BC cases in TCGA training set into the low- and high-risk groups. We confirmed that the high-risk group had a significantly higher percentage of patients with dead status in comparison to the low-risk group. The expression features of the 9 selected glycosyltransferase genes are shown in the heatmap F.Based on TCGA datasets, the univariate and multivariate regression analyses revealed that the risk score was correlated with the prognosis (p < 0.05)  . The lowTo validate the correlation between the expression of glycosyltransferase genes and tumor-infiltrating immune cells (TIICs), a clinical cohort comprised of 20 BC patients under different clinical stages was involved. The expression level of 9 glycosyltransferase genes detected by qRT-PCR was used to calculate the risk scores of the 20 patients  to perform lectin blots. ConA binds to α-linked mannose (α-Man). PHA-L could specifically bind β1,6-GlcNAc branched N-glycan. PHA-E binds to biantennary galactosylated N-glycan with bisecting N-acetylglucosamine. Significantly increased intensities of ConA, PHA-L, and PHA-E revealed the higher expression of N-glycans in the high-risk group  . FurtherNext, we also detected the clinicopathological features and prognosis of BC, including the state of infiltration of immune cells, CNVs, and TMB. Glycosylation plays an increasingly pivotal role in regulating immune-related function and antitumor immunity. Xu et al. confirmed that FUT7, IL4I1, and ITGB7 could remodel the glucose metabolism to strengthen the immunotherapy effect . The pivST3Gal1 is an important sialyltransferase that catalyzes α2,3-linked sialic acid to galactose-containing substrates. The ST3GAL1 upregulation is an event that indicates a worse prognosis in patients and is associated with chemoresistance . Chong eN-glycosyltransferase STT3A to catalyze glycosylation of PD-L1 and maintain PD-L1 stability in hepatocellular carcinoma  can be found in the article/The studies involving human participants were reviewed and approved by Tongji Hospital affiliated with Tongji Medical College. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.YW, JZ, and QZ conceived the project and supervised all experiments. WL, HY, and YT conducted experiments and analyzed the data. HY and MW wrote and revised the manuscript. MH provided support for experimental techniques. MW edited and revised the manuscript, including figures and tables. All authors have reviewed the manuscript and all approved of the final version.This work was supported by China Guanghua Science and Technology Foundation (grant number 2019JZXM001) and Wuhan Science and Technology Bureau (grant number 2020020601012241).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Epithelial ovarian cancer is usually diagnosed in the advanced stages. To choose the best therapeutic approach, an accurate preoperative assessment of the tumour extent is crucial. This study aimed to determine whether the peritoneal cancer index (PCI), the amount of ascites, and the presence of cardiophrenic nodes (CPLNs) visualized by computed tomography (CT) can assess the tumour extent (S-PCI) and residual disease (RD) for advanced ovarian cancer (AOC) patients treated with upfront surgery.In total, 118 AOC cases were included between January 2016 and December 2018 at Skåne University Hospital, Lund, Sweden. Linear regression and interclass correlation (ICC) analyses were used to determine the relationship between CT-PCI and S-PCI. The patients were stratified in complete cytoreductive surgery (CCS) with no RD or to non-CCS with RD of any size. The amount of ascites on CT (CT-ascites), CA-125 and the presence of radiological enlarged CPLNs (CT-CPLN) were analysed to evaluate their impact on estimating RD.p < 0.001). The risk of RD was also related to CT-PCI (OR 1.069 (1.009–1.131), p < 0.023) with a cut-off of 21 for CT-PCI . The sensitivity, specificity, positive predictive value and negative predictive value were 58.5, 70.3, 52.2 and 75.4%, respectively. CT-ascites above 1000 ml predicted RD (OR 3.510 (1.298–9.491) p < 0.013).CT-PCI correlated well with S-PCI (0.397; 95% CI 0.252–0.541; CT is a reliable tool to assess the extent of the disease in advanced ovarian cancer. Higher CT-PCI scores and large volumes of ascites estimated on CT predicted RD of any size. Epithelial ovarian cancer is the gynaecological malignancy with the highest mortality rate, with a five-year survival rate below 45% , 2. MoreThe standard treatment for AOC is primary debulking surgery (PDS) followed by platinum-based postoperative chemotherapy . Since sInvestigators have tried to find the optimal benefit of preoperative CT scans to identify radiological predictors of tumour extent . Along wCA-125 is a high-molecular-weight glycoprotein expressed in a large number of diseases and by a large proportion of epithelial ovarian cancer cases, with poor specificity and sensitivity . SeveralThis study aims to investigate whether preoperative imaging  is predictive of tumour extent (S-PCI) and residual disease of any size.A total of 194 patients with AOC (FIGO stage III-IV) were diagnosed and treated from January 2016 to December 2018 at Skåne University Hospital, a tertiary centre for gynaecologic cancer treatment in Southern Sweden. Patient data were collected retrospectively from the patients’ medical records. Patients receiving neoadjuvant chemotherapy with interval debulking surgery  or with palliative chemotherapy only  were excluded. Twenty-eight patients were excluded due to insufficient data. In total, 118 patients who were deemed suitable for upfront extensive surgery were included in the study.Clinical data on tumour stage (FIGO), histology type, age, performance status (ECOG: Eastern Cooperative Oncology Group) and CA-125 were collected.The clinical characteristics of all patients included in the study are shown in Table To ensure the accuracy of the surgical documentation and S-PCI, both procedures were performed by the same surgeon for a series of 25 patients in the cohort. The author calculated the S-PCI score for the 25 surgical records, which was compared with the original S-PCI score calculated by the surgeon for each case. The mean and standard deviation (SD) of the original S-PCI was 17.07 ± 9.41, and the newly calculated S-PCI was 16.23 ± 9.03. As the data were normally distributed, Student’s t-test was used to confirm a very good correlation with the original measurement. Based on these findings, the S-PCI in the remaining cases was assessed according to the original surgical reports. The PCI score developed by Sugarbaker (1998) was used to quantify both CT-PCI and S-PCI [To evaluate the possible effect of time on the relationship between CT-PCI and surgery (S-PCI), CT-PCI was assessed within 20 days, between 21 and 40 days and beyond 40 days. The 20-day interval was chosen as a reference according to the actual standardized cancer care pathway in Sweden, which requires a maximum of 24 days until the start of treatment .The volume of the ascites at the surgery was categorized into three groups . Twenty-three of 118 patients underwent paracentesis because of abdominal swelling or tightness. Data from 10 patients were incomplete.Operability and resectability were evaluated for each patient according to age, medical history, symptoms, medication, social and nutritional status and feasibility of tumour debulking according to preoperative CT of the abdomen and thorax. All therapeutic decisions were made for each patient by a multidisciplinary team (MDT) consisting of imaging specialists, gynaecologic oncologists, medical oncologists and pathologists. Only patients intended for upfront surgery were included in the study. The surgical outcome was indicated by the amount of residual disease after the surgery. The surgical outcome was stratified into CCS with no residual disease at the end of the surgery and non-CCS, summarizing patients with residual disease of any size.All eligible patients underwent CT in the supine position with intravenous and oral contrast. Digital CT images were obtained by convention reformatted in the coronal and sagittal planes. CT-PCI was retrospectively scored using the Sugarbaker classification  by one ot test and the Mann–Whitney U test were used to evaluate the associations of S-PCI and CT-PCI with clinical factors.The Stata SE  and IBM SPSS Statistics 25 were used for all statistical analyses. For descriptive data, including means, medians and percentages, standard analyses were used. The normality of the data was tested using the one-sample Kolmogorov–Smirnov test. FIGO stage, ECOG score, surgical outcome, and presence of CPLNs were dichotomized and analysed in terms of numbers and percentages. Student’s Linear regression and interclass correlation (ICC) analyses were used to characterize the agreement between CT-PCI (continuous) and S-PCI (continuous). The data were adjusted for CA-125 level (continuous), ascites  and the number of days between the CT examination and surgery . An ICC analysis was used to compare S-ascites and CT-ascites , and weighted kappa and percent agreement were calculated. The kappa value was used to compare the agreement between those two groups . A kappa value of 1.00 is reflective of perfect agreement, 0 indicates no agreement, 0.81–1.00 shows very good agreement, and 0.61–0.80 shows good agreement.Linear and logistic regression analyses were used to determine the relationships between CT-PCI, S-PCI and surgical outcome. Analyses were adjusted for different variables . Some data were log transformed due to an asymmetric distribution (CA 125).The receiver operating characteristic curve (ROC) was used to calculate a cut-off for CCS for both CT-PCI and S-PCI.p value of < 0.05 was considered significant.Linear and logistic regression analyses were used to determine the impact of positive radiological CPLNs on the surgical outcome. We compared the presence or absence of CPLNs, dichotomized and continuous. The results are presented as odds ratios (ORs) with 95% confidence intervals (CIs). A  p < 0.001))  (0.511 (95% CI 0.387–0.639), p < 0.001) Fig. . Statist)) Table .Fig. 2LiWhen the ICC was used to compare CT-PCI and S-PCI divided into three groups , an agreement of 79% was found (0.580 (95% CI 0.442–0.0.991)).p = 0.711).No difference between the tumour extent of the different FIGO stages was found; the mean S-PCI was 16.31 for FIGO stage 3 and was 16.96 for FIGO stage IV (p < 0.038) (Table The amount of ascites was positively correlated with the S-PCI in both the crude and adjusted data (for ascites volume > 1000 ml: 4.390 (95% CI 1.027–7.753) p < 0.001), but when the data were adjusted for CT-PCI, ascites, and days between CT examination and surgery, no significant association between CA-125 and S-PCI was found (Table The CA-125 level was related to tumour extent in the crude data (1.439 (0.665–2.212), p < 0.001).The results from logistic regression models showed a significant association between CT-PCI and residual disease (OR 1.088 (1.037–1.142), p < 0.023) (Table p = 0.000) and generating a cut-off value of 21 for CT-PCI , 2, p < 0.To identify a reason for unresectability, a group of 19 patients with residual disease ≥ 10 mm was analysed separately. The site of the residual disease and other indicated reasons for terminating the surgery were small intestinal carcinomatosis in 12 cases, and in 9 (75%) of these, intestinal carcinomatosis referred to abdominal regions from 9 to 12  on the CT scan.p < 0.013)) , n = 60) of the patients exhibited CPLNs ≥ 5 mm in the short axis of the CT scan. In 46% (n = 56) of the patients, the dominant lymph node was located on the anterior side of the cardiophrenic space, whereas in 22% (n = 27) of the patients, it was located on the posterior side. Twenty-three patients had enlarged lymph nodes on both the anterior and posterior sides. A median of 1.17 (range 0–6) enlarged CPLNs was detected. The presence of CPLNs was not correlated with the amount of residual disease at the end of the surgery in any of the analyses , although not significant in either unadjusted or adjusted results for CT-PCI, CPLN, ascites and days between diagnosis and surgery, The preoperative estimation of tumour extent and its resectability are both of great importance to surgical outcome in AOC. In this study, both CT-PCI and CT ascites were correlated with the S-PCI, showing that CT might be a reliable tool in the preoperative assessment of tumour extent. The results are in accordance with previous studies on the estimation of tumour extent in ovarian cancer , 21. In The S-PCI was related to the time interval between the CT scan and surgery. Patients with a longer time interval than the reference of 20 days had higher S-PCI scores. This can be explained by tumour progression or a higher tumour load from the beginning, leading to impaired nutritional status that requires a longer preoperative rehabilitation time and a prolonged time between the CT scan and surgery. A future recommendation for the time lapse between CT and surgery might be warranted.A good correlation was also found between the preoperative estimation of ascites volume and S-PCI, indicating that patients with CT ascites above 1000 ml had a 4.4-times higher risk of an increased S-PCI score, indicating a more pronounced tumour extent.Residual disease after primary debulking surgery is one of the most important prognostic factors for survival in AOC . This stLlueca et al.  tried toCT may both overestimate and underestimate the true peritoneal carcinomatosis amount . TechnicSome studies have analysed separate regions and found that PCI in certain areas is more related to unresectability. Abdominal regions 9–12, corresponding to the small intestine, were significantly more predictive of residual disease than the entire PCI . The samThe quantification of ascites has, to our knowledge, been sparsely evaluated in preoperative CT scans in patients with AOC. In this study, the CT-based evaluation of ascites volume correlated with the ascites volume determined intraoperatively. When ascites is quantified intraoperatively, large volumes correlate with unresectability and worse PFS and OS , 33. MasCPLNs in AOC are associated with carcinomatosis in the upper abdomen, diaphragmatic carcinomatosis, and a worse prognosis regardless of surgical removal during cytoreductive surgery. Of the 350 patients analysed by Prader et al. , almost The relationship between CA-125 level and surgical outcome is controversial. Many studies have found a correlation between CA-125 > 500 U/mL and suboptimal cytoreduction , 34, 35.The strength of this study is that it was conducted in a single tertiary centre with a high CCS rate and highly specialized radiology department, both of which increase the accuracy of CT-PCI and S-PCI scoring. The fact that the patients’ clinical records, including their histopathological records, are all connected to one entity, thus making it possible to verify PCI as true carcinosis, can be considered a strength as well.Interreader variability was not addressed which is a limitation of the study. However, the image evaluation may be seen as a consensus evaluation made by the referring hospital radiologist and the multidisciplinary conference radiologist. Another limitation of this study is the retrospective nature of PCI documentation and the relatively small cohort of patients.Numerical estimation of the tumour extent by CT is feasible in AOC but may require a skilled radiologist. Together with a high volume of ascites, as shown in this study, the CT-PCI might be used as an indicator for residual disease in upfront surgery for AOC. Further prospective studies are needed.


Dulaglutide is associated with improved cardiovascular and kidney outcomes and can be a good therapeutic option for patients with type 2 diabetes with chronic kidney disease (CKD). In this study, the effects of dulaglutide on glucose-lowering efficacy and changes in renal function were analyzed.This retrospective study involved 197 patients with type 2 diabetes with mild-to-severe CKD treated with dulaglutide for at least 3 months between January 2017 and December 2020 at two tertiary hospitals in Korea. Changes in the creatinine-based estimated glomerular filtration rate (eGFR) and HbA1c were compared before and after the use of dulaglutide in each patient.2) in CKD 2, 3a, 3b, and 4 were 94 (75.0 ± 8.5), 46 (54.8 ± 6.3), 31 (38.8 ± 4.4), and 26 (22.5 ± 5.4), respectively. Mean HbA1c level and body mass index (BMI) at the initiation of dulaglutide were 8.9% ± 1.4% and 29.1 ± 3.6 kg/m2, the median duration of the use of dulaglutide was 16 months. The use of dulaglutide was associated with a mean decrease in HbA1c by 0.9% ± 1.5% and the glucose-lowering efficacy was similar across all stages of CKD. Also, it was associated with a reduced decline in the eGFR; the mean eGFR change after the use of dulaglutide was –0.76 mL/min/1.73 m2 per year, whereas it was –2.41 mL/min/1.73 m2 before use . The difference was more pronounced in patients with an eGFR < 60 mL/min/1.73 m2. Subgroup analysis showed that the renal protective effect was better in patients with proteinuria, age ≤ 65 years, and HbA1c < 9.0%, but showed no association with BMI.The number of patients and mean eGFR (mL/min/1.73 mThe use of dulaglutide provided adequate glycemic control irrespective of CKD stage and was associated with a reduced decline in the eGFR in the CKD population. In general, while recent guidelines advocate the use of sodium-glucose co-transporter 2 (SGLT2) inhibitors with metformin as first-line therapy, neither is recommended for initiation in advanced CKD . . 3]. Hownced CKD –6. In adnced CKD , 8.GLP-1 is secreted by the L-cells of the small intestine in response to glucose ingestion . Augment2. While most incretin-mimetics are ‘short-acting’ drugs, human GLP-1RAs are called ‘long-acting’ since specific molecular characteristics, such as the covalent bond with albumin , with the Fc portion of human immunoglobulin G4 (dulaglutide), or with specific fatty acids (liraglutide) give them a long half-life and prevent their elimination by the kidneys. Therefore, contrary to short-acting GLP-1RAs, human GLP-1 analogues induce a more marked reduction in HbA1c and fasting blood sugar and decrease the incidence of side effects https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  23 Jul 2022Dear Editor,Dear Reviewers,We greatly appreciate the opportunity to revise our paper in light of the reviewers’ comments and resubmit it for publication in PLOS ONE.Manuscript Title: "Effect of once-weekly dulaglutide on renal function in patients with chronic kidney disease” Manuscript ID: PONE-D-22-17686We made our best efforts in order to make the requested revisions in light of the editor and reviewer’s comments. Below you can find an itemized, point-by-point detailed response to all the questions and comments of the reviewers.We hope our paper is now suitable for publication in PLOS ONE in its present form and we are now resubmitting it to your attention.REVIEWERS’ Comments:Reviewer #1 Comments:In this manuscript, the authors investigated renoprotective effects of dulaglutide in patients with type 2 diabetes (T2D) and CKD. They performed retrospective analysis and compared eGFR changes a year between before and after starting dulaglutide. They observed that dulaglutide use was associated with improvement of eGFR decline (-2.41 vs. -0.76 mL/min/1.73m2 per year). The renoprotective effects were observed strongly in patients with eGFR<60 (-3.7 vs. -1.1 mL/min/1.73m2 per year). Dulaglutide use resulted in reductions in HbA1c levels across the renal function.Comment:1) Resolutions of Figures are not enough.Answer:In the revised manuscript, we increased the resolution of each Figure. Thank you.Special Comment:2) What was the dose-response reno-protective effects by dulaglutide? Were any differences in 0.75 mg and 1.5 mg observed?Answer:As you pointed out, it may be needed to compare the efficacy between 0.75mg and 1.5mg of dulaglutide. In clinical practice, however, 0.75mg/week of dulaglutide is usually used during the first 2 weeks to monitor for side effects, after which the dose is increased to 1.5mg/week. So, in this study, we could not directly compare the efficacy between 0.75mg and 1.5mg. But according to the AWARD-7, either dulaglutide 1.5mg or 0.75 mg was associated with a significantly smaller decline of eGFR from baseline compared with insulin glargine. We added this in the limitation of the revised manuscript. Comment:3) Changes in albuminuria by dulaglutide use should be shown.Answer:Thank you for your comments. In the revised manuscript, we added the data comparing changes in urinary albumin before and after the use of dulaglutide . As shown in the figure below, the use of dulaglutide significantly reduced the incidence rate of progression from microalbuminuria to proteinuria. Before the use of dulaglutide, the progression of microalbuminuria to proteinuria occurred in 35.7% of the cases, but after the use of the dulaglutide, the incidence of proteinuria development from microalbuminuria occurred only in 7.0%. Whereas, we did not find any difference in the incidence of new-onset microalbuminuria with the use of dulaglutide use. We added this in the RESULT section of the revised manuscript. Comment:4) I wonder if the effects of improvement of glycemic control on eGFR changes were excluded.Answer:Thank you for your kind comments. There was no correlation between HbA1c level after dulaglutide use and eGFR changes . However, as we compared the eGFR changes before and after the use of dulaglutide, we could not predict the eGFR slope with the use of dulaglutide. In this regard, we cannot precisely adjust the effect of glycemic control on the eGFR decline rate. But as shown in Figure 3C, the reduction in the eGFR decrease with the use of dulaglutide was not clear in patients with very high baseline HbA1c levels. We added this in the limitation section of the revised manuscript. Comment:5) It seems that eGFR changes by dulaglutide use were robust compared to previous reports. What is specific reason underlying this observation?Answer:We totally agree with your opinion. We think that the clear difference observed in our study occurred because we compared the mean eGFR changes before and after the use of dulaglutide in each patient. As you know, diabetic kidney disease is a very heterogeneous disease entity, therefore, the renal progression rate differs greatly depending on the clinical circumstances. So, rather than analyzing all DKD patients with very diverse clinical manifestations, we compared before- and after-changes in each patient, so the result seems to be much more robust compared to other reports.Comment:6) Non-users of dulaglutide should be included as control.Answer:Thank you for your comments. As you mentioned, if this study evaluated the efficacy of dulaglutide in DKD patients, we definitely need non-users as control. But in this study, we compared the changes before and after the use of dulaglutide in each patient, so we did not include non-users. We added this in the DISCUSSION section in the revised manuscript. Reviewer #2 Comments:General Comment:The authors describe the effects of dulaglutide in a retrospective study of 197 type-2 diabetic patients with different CKD stages. The paper is well written and the limitations of the study are clearly considered in the discussion.I have no MAJOR CONCERNS.- As MINOR COMMENTS I suggest the authors to consider some changes:- The authors are using the term "macroalbumini¡uria!. Cam I suggest to change to that of "proteinuria " in files 191,193,203,229, 230, 231, 240, 241, 297, 312, 314 and 347 ?-- Key words , file 74: GLP-1 receptor agonists- file 151... weekly.- file 303: Randomized Clinical Tral (RCT).- file 326: body weight (BW).Answer:Thank you for your kind comments. As you recommended, we all changed the “macroalbuminuria” to “proteinuria” in the revised manuscript. Also, all the typographical errors were corrected.Reviewer #3 Comments:Comment:The introduction should be reorganized. In my opinion, comments on the results of clinical trials and meta-analysis (page 6 - lines 124-127) should be postponed for discussion. In this section, I would better describe the following aspects:https://diabetesjournals.org/care/issue/45/Supplement_1)Page 5 – lines 104-105: “…while recent guidelines advocate the use of sodium-glucose co-transporter 2 105 (SGLT2) inhibitors with metformin as first-line therapy…”. I suggest inserting a bibliographic note in this sentence referring to the latest guidelines of the American Diabetes Association (available at the site doi.org/10.1093/ckj/sfac069).Page 5 – lines 117-119: regarding the safety profile and pharmacokinetics of GLP1-RAs, I believe it is necessary to make a brief clarification about the different pharmacological properties of incretion-mimetics and human GLP-1 analogues , which have only 52% structural analogy to endogenous GLP-1 and thereby have great immunogenic power with potential development of inactivating antibodies, human GLP-1 analogues  have low immunogenic power due to a high structural analogy with endogenous GLP-1. Another fascinating aspect of the use of GLP-1RAs is that they can be initiated in patients with an eGFR as low as 15 mL/min/1.73 m2. While most incretin-mimetics are ‘short-acting’ drugs, human GLP-1RAs are called ‘long-acting’ since specific molecular characteristics, such as the covalent bond with albumin , with the Fc portion of human immunoglobulin G4 (dulaglutide), or with specific fatty acids (liraglutide) give them a long half-life and prevent their elimination by the kidneys. Therefore, contrary to short-acting GLP-1RAs, human GLP-1 analogues induce a more marked reduction in HbA1c and fasting blood sugar and decrease the incidence of side effects”. Also, we added the recent ADA guideline as new reference.Comment:Materials and methodsPage 7 - lines 162-164: please define more clearly the definitions of micro- and macroalbuminuria.Answer:To avoid confusion, the term “macroalbuminuria” was deleted from the revised manuscript and changed to “proteinuria”. Microalbuminuria was defined as a UACR 30–300 mg/g. Patients with a UACR >300mg/g or UPCR >0.2 g/g were regarded as having proteinuria.Comment:Discussiondoi.org/10.1093/ckj/sfac069). Similar conclusions have been reported also in a recent meta-analysis 00203-5.), suggesting a determining role of GLP1-RAs in slowing the progression of renal damage beyond proteinuria. In my opinion, these aspects should be discussed in this section.Pag 13 lines - 294-301: it should be noted that the benefits of dulaglutide on worsening eGFR in the REWIND trial are underestimated. The re-evaluation of the data, conducted with the sensitivity analysis method, has shown that dulaglutide significantly reduces the worsening eGFR when it is defined as a reduction of ≥40% or ≥50%, rather than ≥30% as in the original study design (see Granata et al. https:/Answer:Thank you very much for your kind comments. In the revised manuscript, we corrected the paragraph as follows. “Also in the REWIND trial, which included mostly low-risk patients with a mean eGFR of 75 mL/min/1.73 m2, composite renal outcomes, mainly with the incidence of de novo macroalbuminuria, occurred significantly less frequently in the dulaglutide group compared to placebo.[25] In addition, recently, the re-evaluation of the REWIND data with sensitivity analysis revealed that dulaglutide significantly reduces the worsening eGFR when it is defined as a reduction of ≥40% or ≥50%, rather than ≥30% in the original analysis.” Also, some more references were added.Thank you so much for your kind comments.Sincerely,Jwa Kyung Kim, MD, PhDHallym University Sacred Heart Hospital, KoreaAttachmentResponse to Reviewers.docSubmitted filename: Click here for additional data file. 1 Aug 2022Effect of once-weekly dulaglutide on renal function in patients with chronic kidney diseasePONE-D-22-17686R1Dear Dr. Kim,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact Kind regards,Tomislav BulumAcademic EditorPLOS ONEAdditional Editor Comments:In revised manuscript term macroalbuminuria is still present in some part of Results and Discussion section. Please change to proteinuria. 4 Aug 2022PONE-D-22-17686R1 Effect of once-weekly dulaglutide on renal function in patients with chronic kidney disease Dear Dr. Kim:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Tomislav Bulum Academic EditorPLOS ONE